Abnormal neuronal activity in the brain leads to epileptic seizures that, when repeated or prolonged, can cause neuronal damage resulting in delayed psychomotor development and intellectual disability. Most genetic variants associated with epilepsy are in genes encoding ion channels, including potassium channels that regulate neuronal excitability such as IKM channels. Inherited mutations in the IKM channel cause a wide spectrum of early-onset epileptic disorders. The long-term goal of this research program is to understand the mechanisms by which the wt IKM channel work, how epilepsy-causing mutations lead to dysfunction of IKM channels and to design drugs that correct IKM dysfunction. The objective of this application is to determine how epilepsy-inducing mutations in the IKM subunits KCNQ2 and KCNQ3 cause channel malfunction. Because polyunsaturated fatty acids (PUFAs) have been shown to alleviate the symptoms of intractable epileptic seizures, we will investigate the mechanisms by which these compounds reverse channel malfunction and therefore improve neuronal function. The overarching hypothesis is that that epilepsy-causing mutations in KCNQ channels affect voltage sensor movement and that PUFAs can restore normal voltage dependence of voltage sensor movement in mutated KCNQ channels. The rationale for the proposed research is that understanding the molecular basis by which different mutations in the IKM channel are linked to epilepsy will not only help explain epilepsy pathogenesis but also provide clues for intervention strategies. Guided by preliminary data, we will test our hypothesis by pursuing three specific aims: (1) determine the mechanisms by which epilepsy-causing mutations affect IKM channels function. (2) determine how PUFAs affect voltage sensor and gate movements of IKM channels bearing epilepsy-associated mutations and to identify which PUFA variants restores channel function, and (3) determine whether PUFAs reduce hyperexcitability on neurons bearing epilepsy-causing mutations in IKM channels. Under the first Aim, we will combine cysteine accessibility and VCF approaches to simultaneously measure voltage sensor movement and gate opening in the wt IKM and a set of epilepsy-associated mutants. This will allow us to determine how mutations affect the movement of the voltage sensor and the activation gate in KCNQ2 and KCNQ3 channels. We will also incorporate unnatural amino acids (UUAs) into mutated channels (UUAs mutagenesis) to further map the molecular determinants of channel dysfunction. Under Aim 2, we will test PUFA variants with different chain lengths, different acyl chains and different types of polar head groups to determine the molecular mechanism of PUFA?s effects on these mutations. Under the third Aim, we will test PUFA variants that can correct channel function and restore activity in iPSC-derived cortical neurons bearing epilepsy-associated mutations in KCNQ2 and/or KCNQ3. The proposed research is significant because the anticipated results will provide the mechanistic basis for how mutations cause IKM channel defects and will show proof-of-concept that PUFAs can act as antiepileptic drugs.